Assay for Detecting TH1 and TH2 Cell Populations

ABSTRACT

The present disclosure relates to a method for detecting T helper cell or CTL subpopulations in a subject affected by disease or disorder having an immune component. The methods are also useful for determining efficacy of a treatment of the disease or disorder by detecting skewing of the T helper cells and CTLs in a therapeutic or adverse direction.

FIELD OF THE INVENTION

The present disclosure relates to a method for detecting T helper (Th)cell and polyfunctional cytotoxic T-cells (CTL) subpopulations in in asubject affected by disease or disorder having an immune component. Themethods are also useful for determining efficacy of a treatment of thedisease or disorder by detecting skewing of the T helper or CTL cells ina therapeutic or adverse direction.

BACKGROUND OF THE INVENTION

T-cell subsets include helper T-cells (CD4+) and cytotoxic T-cells (CD8+CTL). CD4+ helper T cells can generally be broken down into severaldifferent subtypes. Commonly divided populations include T helper 1(Th1) and T helper 2 (Th2) cells which have been found to play distinctroles in mediating immune-related diseases and disorders. Th1 and Th2cells can be distinguished based on their cytokine expression profile.Th1 cells typically express interferon-gamma (IFN-g, IFN-γ),lymphotoxin, (LT), and can also secrete interleukin-2 (IL-2) and tumornecrosis factor-alpha (TNF-a, TNF-α) (Zhu et al., Annu Rev Immunol. 28:445-489, 2010). Th2 cells are characterized by production of IL-4, IL-5and IL-13, and do not secrete IFN-γ or lymphotoxin. Some Th2 cells havebeen found to produce TNF-α and IL-9 (Zhu, supra). Th17 cells have alsorecently been determined to be a different subset of T helper cells.Th17 cells secrete IL-17A, IL-17F, and IL-22 cytokines, and can alsoproduce IL-21. The dysregulation of the ratio of Th1 to Th2 cells, aswell as Th17 cells, has been associated with certain disease states,including autoimmune diseases, allergic reactions and cancers. Twoadditional CD4+ T-cell subsets that can be identified by cytokineproduction include Treg (IL-10) and follicular helper T cells (Tfh)(IL-21).

Polyfunctional, CD8+ CTL are important effector cells that provideprotection from intracellular pathogens and tumors wherepolyfunctionality is defined as production of multiple cytokinesincluding (e.g. IFN-g, TNF-a and/or IL-2). In cancer subjects thesepolyfunctional T-cell responses have been shown to be repressed. Severalnew treatments (e.g. anti- CTLA4, anti-PD1) have been shown reversefunctional suppression of CTL and combination with TVEC (talimogenelaherparepvec) holds further promise for increasing the number offunctional, activated CTL that can contribute to meaningful anti-tumorresponses. Additional T cell subsets include natural killer T cells(NKT) and gamma delta T cells (gdT).

Determining levels of T cells in vivo and determining the levels ofcytokines produced by these cells can be difficult. Recent advances inlaboratory techniques have provided several methods for determining Thelper subtypes and cytokine levels, including ELISA, ELISPOT, and flowcytometry assays, including intracellular cytokine staining assays.Despite the advancement in methodology, it is still difficult toidentify particular T cell subtypes and their cytokine profiles in apatient with a disease or disorder because the levels of theseparticular cell types and cytokines in a given patient sample can be lowto undetectable.

A common technique to identify and separate one cell type from anotheris flow cytometry using labeled dyes, e.g., in a fluorescence activatedcells sorter (FACS). Methods of carrying out flow cytometry arediscussed, for example, in U.S. Pat. Nos. 8,389,291, 7,932,503,7,012,689, and 6,287,791.

In addition to cytokines, combinations of cell surface markers can alsobe used to classify T-cells by subset (e.g. Th2 cells express CRTH2) oractivation status (e.g. activated CTL express HLA-DR) by FACS.

Previous studies have undertaken experiments to analyze T helper cellprofiles in response to stimuli. International Patent Publication WO1997/026883 describes intracellular cytokine staining for IL-2 only onPBMC stimulated in vitro with PMA/ionomycin with or without ribavirin®.The results provided describe a putative pharmacological effect based onin vitro data.

U.S. Pat. No. 6,039,969 describes animal model data demonstrating that aclass of compounds including the drug imiquimod can skew the immuneresponse away from Th2, based primarily on secreted cytokine results.Human clinical data and intracellular cytokine staining were notdisclosed.

U.S. Patent Publication 2001/0006789 describes a method used to identifyantigen specific T-cells. Measurement of pharmacological effects onantigen specific T-cells is not demonstrated.

International Patent Publication WO 2000/024245 describes a method fortargeting NFATp and/or NFAT4 to modulate Th2 cells. WO 2002/089832relates to a combination of cytokine+SDF-1a to skew the Th1/Th2 ratio.In vitro data, including intracellular cytokine staining specific forIFN-g and IL-4, using cultured cord blood T-cells are presented. Humanclinical data demonstration is not included.

Lore et al. (J Immunol 171:4320-28, 2003) describes analysis of CMV- andHIV-specific CD4+ and CD8+ T cells using a method in which levels of theTh1-specific cytokines IFN-g, TNF-a and IL-2 are measured in a singlesample by flow cytometric analysis. The Th1 cytokines are detected usingcytokine-specific antibodies, each of which is labeled with the samefluorophore. This method does not determine the ratio of Th1 to Th2cells.

Ludviksson et. al. (J Immunol 160:3602-3609, 1998) describes associationof Wegener's Granulomatosis with HLA-DR+CD4+ cells exhibiting unbalancedTh1 cell cytokine pattern and reversal with IL-10. Wang et. al. (Int Imm21:1065-1077, 2009) demonstrate that PD1 blockade reverses functionalsuppression of CTL in melanoma setting. Sfanos et. al. (Clin Cancer Res14:3254-3261, 2008) demonstrate skewing of tumor infiltratinglymphocytes to Th17 and Treg subsets in prostate cancer.

The present disclosure is directed to methods for identifying anddifferentiating a population of T helper or CTL cells in a subjecthaving a disease or disorder with an immune component. The presenttechnique can be carried out before and after treatment with atherapeutic agent to improve treatment regimens for patients receivingtherapy.

SUMMARY OF THE INVENTION

The present disclosure provides a method for measuring changes in T cellsubset populations in a subject having a disease or disorder orsubsequent to a treatment regimen. The method is useful to determineefficacy of a treatment regimen and assist in designing an optimaltreatment regimen for a patient.

The present disclosure provides a method for detecting a ratio of Thelper 2 (Th2) and T helper 1 (Th1) cells in a sample from a patientcomprising measuring levels of Th2-specific and Th1-specific cytokines,the method comprising a) contacting the sample with i) two or morespecific binding agents that bind two or more Th2 cytokines, wherein thetwo or more specific binding agents for the Th2-specific cytokines arelabeled with a first fluorophore; and ii) at least one specific bindingagent that binds a Th1 cytokine, wherein the specific binding agent forthe Th1 cytokine is labeled with a second fluorophore that is differentfrom the first fluorophore; b) measuring levels of the first and secondfluorophores in the sample and designating the cell as a Th1 or Th2 cellbased on the level of Th1-specific and Th2-specific fluorophoredetected; and c) determining the ratio of Th2 to Th1 cells in a sample.

In various embodiments, the method further comprises determining theratio of Th1, Th2 and Th17 cells in the sample, comprising a) contactingthe sample with iii) at least one specific binding agent that binds aTh17 cytokine, wherein the specific binding agent for the Th17 cytokineis labeled with a second fluorophore that is different from the firstfluorophore or labeled with a third fluorophore; b) measuring levels ofthe first and second (and/or third) fluorophores in the sample anddesignating the cell as a Th1, Th2 or Th17 cell based on the level ofTh1-specific, Th2-specific or Th17-specific fluorophore detected; and c)determining the ratio of Th1 to Th2 to Th17 cells in a sample.

In various embodiments, the disclosure provides a method for detecting aratio of T helper 1 (Th1) and T helper 17 (Th17) cells in a sample froma patient comprising measuring levels of Th1-specific and Th17-specificcytokines, the method comprising: a) contacting the sample with i) twoor more specific binding agents that bind two or more Th17 cytokines,wherein the two or more specific binding agents for the Th17-specificcytokines are labeled with a first fluorophore; and ii) at least onespecific binding agent that binds a Th1 cytokine, wherein the specificbinding agent for the Th1 cytokine is labeled with a second fluorophorethat is different from the first fluorophore; b) measuring levels of thefirst and second fluorophores in the sample and designating the cell asa Th1 or Th17 cell based on the level of Th1-specific and Th17-specificfluorophore detected; and c) determining the ratio of Th1 to Th17 cellsin a sample.

In various embodiments, the disclosure provides a method for detecting aratio of T helper 2 (Th2) and T helper 17 (Th17) cells in a sample froma patient comprising measuring levels of Th2-specific and Th17-specificcytokines, the method comprising: a) contacting the sample with i) twoor more specific binding agents that bind two or more Th2 cytokines,wherein the two or more specific binding agents for the Th2-specificcytokines are labeled with a first fluorophore; and ii) at least onespecific binding agent that binds a Th17 cytokine, wherein the specificbinding agent for the Th17 cytokine is labeled with a second fluorophorethat is different from the first fluorophore; b) measuring levels of thefirst and second fluorophores in the sample and designating the cell asa Th2 or Th17 cell based on the level of Th2-specific and Th17-specificfluorophore detected; and c) determining the ratio of Th2 to Th17 cellsin a sample.

In certain embodiments, it is also contemplated that the ratio of Th1 toTh2 cells is calculated using the present method, and this ratio used todetermine treatment efficacy and potential change in treatment regimenfor a patient. Similarly, for the methods described herein, it isspecifically contemplated that the converse ratio (e.g., Th17/Th1,Th17/Th2 or any combination ratio for Th1, Th2 and Th17) may bedetermined and a treatment decision made also based on the ratio ofcells determined.

In various embodiments, the Th2 cytokines are selected from the groupconsisting of IL-4, IL-5 and IL-13. In various embodiments, the Th1cytokines are selected from the group consisting of interferon gamma(IFN-g), tumor necrosis factor alpha (TNF-a) and IL-2.

In various embodiments, the Th17 cytokines are selected from the groupconsisting of IL-17A, IL-17F, IFN-g and IL-22.

In various embodiments, the specific binding agent is an antibodyspecific for a Th1-specific, Th2-specific or Th17-specific cytokine.

In various embodiments, the method further comprises a step ofadministering a therapeutic agent, altering a dose regimen of atherapeutic agent or maintaining a dose regimen of a therapeutic agentin view of the ratio of T cell subsets detected by the method, whereindetection of a particular ratio indicates the treatment regimen to beadministered.

In various embodiments, the patient is suffering from a disease ordisorder selected from the group consisting of asthma, allergicrhinosinusitis, allergic conjunctivitis, atopic dermatitis, fibroticdisorders, Systemic Lupus Erythematosus (SLE), multiple sclerosis andcancers. Additional disorders contemplated in the present methods arediscussed in greater detail in the Detailed Description.

In various embodiments, the sample is obtained before and/or aftertreatment with a therapeutic agent. In certain embodiments, the sampleis whole blood, peripheral blood mononuclear cells, cerebrospinal fluid,bronchioalveolar lavage, nasal lavage, induced sputum or a biopsy fromthe patient.

In various embodiments, the therapeutic agent is an anti-TSLP antibody.

In various embodiments, the first fluorophore is phycoerythrin (PE) andthe second fluorophore is fluorescein isothiocyanate (FITC). Additionalfluorophores contemplated for use in the method include, but are notlimited to, Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, AlexaFluor® 488, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, AlexaFluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, AlexaFluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, AlexaFluor® 680, Alexa Fluor® 700, BODIPY FL, BODIPY 630/650, Cy2, Cy3, Cy3B,Cy3.5, CyS, Cy5.5, Cy7, ECD, FITC, FluorX®, Cascade® Blue, PacificBlue®, Pacific Green®, Pacific Orange®, eFluor® 450, eFluor® 605NC,eFluor® 625NC, eFluor® 650NC, eFluor® 660, eFluor® 710, BrilliantViolet™ (BV) fluorophores BV421, BV510, BV570, BV 605, BV650, BDHorizon™ V450, BD Horizon™ V500, Texas Red, rhodamine, cyanine,phycoerythrin (PE), phycocyanin, allophycocyanin (APC), o-phthaldehyde,fluorescamine, Oregon Green® 488, PE-APC, PE-Cy5, PerCP, PE-TR,rhodamine green and rhodol green, and tandem dyes thereof.

In one embodiment the cytokines are contacted with the specific bindingagent intracellularly. In certain embodiments, the cytokines arecontacted with the specific binding agents extracellularly. In certainembodiments, the cytokines are secreted from the cells in vitro or inare detectable in fluid samples.

In various embodiments, the disclosure provides a method for identifyinga sub-population of asthma patients responsive to treatment with atherapeutic agent comprising measuring the baseline ratio of T helper 2(Th2) and T helper 1 (Th1) cells in a patient sample or changes in theratio after administration of the therapeutic agent, the methodcomprising: a) contacting the sample with i) two or more specificbinding agents that bind two or more Th2-specific cytokines, wherein thetwo or more specific binding agents for the Th2-specific cytokines arelabeled with a first fluorophore; and ii) at least one specific bindingagent that binds a Th1-specific cytokine, wherein the specific bindingagent for the Th1-specific cytokine is labeled with a second fluorophorethat is different from the first fluorophore; b) measuring levels of thefirst and second fluorophores in the sample and designating the cell asa Th1 or Th2 cell based on the level of Th2-specific and Th1-specificfluorophore detected; c) determining the ratio of Th2 to Th1 cells in asample based on the level of Th2-specific cytokine and Th1-specificcytokine detected, wherein the patient is identified as responsive tothe therapeutic agent if the ratio of Th2 cells/Th1 cells decreases; andd) altering treatment with the therapeutic agent if the patient isdetermined to be non-responsive to the therapeutic agent or maintainingthe dose of therapeutic agent if the patient is determined to beresponsive to treatment with the therapeutic agent. In variousembodiments, the ratio of Th17 cells to Th1 and Th2 cells in the asthmapatient is measured as described herein.

In various embodiments, the therapeutic agent is administered for oneweek, two weeks, three weeks, four weeks, six weeks, two months, threemonths or more prior to obtaining the sample. Additional dosing regimenscontemplated by the method are discussed in greater detail in theDetailed Description.

In various embodiments, the therapeutic agent is an anti-TSLP antibodyor anti-TSLP receptor antibody.

In various embodiments, the asthma patient has atopic asthma, includingmild, moderate, or severe asthma. In certain embodiments, the Th2/Th1ratio in an asthma patient is skewed toward the Th2 phenotype and thisskewing could identify the patient as a candidate for treatment with atherapeutic agent that targets the Th2 pathway. In various embodiments,the patient may be a candidate for Th2 targeted therapy if the ratio ofTh2/Th1 cells is approximately 0.2 or higher.

In one embodiment, the asthma patient is identified as responsive totreatment if the ratio of Th2/Th1 cells decreases by 20%, 30%, 40%, 50%,60% or more. It is contemplated that the degree of responsiveness iscompared to a baseline value obtained before or at certain points duringtreatment. In one embodiment, a Th2/Th1 ratio at which the patient isidentified as responsive to treatment is approximately 0.1 or below.

In various embodiments, the disclosure provides a method of altering thedose regimen of an anti-TSLP agent in treating an immune disordercomprising determining the ratio of Th2/Th1 and/or Th17 cells in asample using the method described herein and altering the dose ofanti-TSLP antibody if the ratio of Th2/Th1 and/or Th17 cells changesduring treatment, wherein the dose of therapeutic is increased if theratio of Th2/Th1 cells is stable or increases indicating skewing towardsTh2 profile (i.e., increasing the proportion of Th2 cells and/ordecreasing the proportion of Th1 cells); and wherein the dose oftherapeutic is decreased if the ratio of Th2/Th1 cells decreasesindicating reduction in Th2 profile (i.e., decreasing the proportion ofTh2 cells and/or increasing the proportion of Th1 cells).

In various embodiments, the immune disorder treated by an anti-TSLP oranti-TSLP receptor agent is an atopic disease or disorder. Examples ofatopic diseases and disorders include, but are not limited to, asthma,allergic rhinosinusitis, allergic conjunctivitis and atopic dermatitis.In certain embodiments, the immune disorder is a fibrotic disorder.

In various embodiments, contemplated herein is a method of altering thedose regimen of an asthma therapeutic comprising determining the ratioof Th2/Th1 and/or Th17 cells in a sample using a method described hereinand altering the dose of asthma therapeutic if the ratio of Th2/Th1and/or Th17 cells changes during treatment, wherein the dose oftherapeutic is increased if the ratio of Th2/Th1 cells is stable orincreases indicating skewing towards Th2 profile; and wherein the doseof therapeutic is decreased if the ratio of Th2/Th1 cells decreasesindicating reduction in Th2 profile.

In certain embodiments, if a high proportion of Th17 cells is detected,the method further comprises administering a therapeutic agent thatinhibits IL-17 activity or the IL-17 pathway.

Also contemplated are methods of detecting CD8+ CTLs. The presentdisclosure provides a method for detecting a subset of CTL, such aspolyfunctional CTLs, the method comprising a) contacting the sample withone or more specific binding agents that bind one or more polyfunctionalCTL cytokines, wherein the one or more specific binding agents for thepolyfunctional CTL cytokines are labeled with a first fluorophore and/orsecond fluorophore and/or third fluorophore; b) measuring levels of thefirst and second and/or third fluorophores in the sample and designatingthe cell as a polyfunctional CTL cell based on the level of specificfluorophore detected; and c) determining the ratio of polyfunctional CTLto other T cells, e.g., Th, Treg, NKT or gdT, in a sample.

In various embodiments, polyfunctional CTL cytokines are selected fromthe group consisting of IFN-g, IL-17, TNF-a and IL-2.

In various embodiments, cell surface markers are used for identificationof T cell subpopulations. Exemplary cell surface markers include, butare not limited to, ST2, CRTH2, and CCR4 for Th2 cells, CXCR3 for Th1cells, and/or CCR6 for Th17 cells. T cell subpopulations are alsodistinguished based on biomarkers indicative of activation state,including PD1, CTLA4, CD4OL, ICOS, OX40, 41BB, TIM-3, GITR, HLA-DR andKi67 of Th1, Th2, and Th17 cells.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations defined by specific paragraphs above. For example,certain aspects of the invention that are described as a genus, and itshould be understood that every member of a genus is, individually, anaspect of the invention. Also, aspects described as a genus or selectinga member of a genus, should be understood to embrace combinations of twoor more members of the genus. Although the applicant(s) invented thefull scope of the invention described herein, the applicants do notintend to patent subject matter described in the prior art work ofothers. Therefore, in the event that statutory prior art within thescope of a paragraph is brought to the attention of the applicant(s) bya Patent Office or other entity or individual, the applicant(s) reservethe right to exercise amendment rights under applicable patent laws toredefine the subject matter of such a paragraph to specifically excludesuch statutory prior art or obvious variations of statutory prior artfrom the scope of such a paragraph. Variations of the invention definedby such amended paragraphs also are intended as aspects of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the allergen-induced percent fall in FEV₁. PercentFEV₁-time curve at screening, day 42 and day 84 are presented Data areshown as mean ±SEM. As compared to placebo, AMG 157 treatmentsignificantly attenuated the late allergen-induced fall in FEV1 at days42 and 84, and attenuated the early allergen-induced fall in FEV₁ atdays 42 and 84. FEV₁ denotes forced expiratory volume in 1 second, *denotes p<0.05 AMG 157 compared to placebo.

FIG. 2 shows the treatment difference estimate (AMG 157 minus placebo)in allergen induced airway responses. Mean estimate was based on ANCOVA.AUC denotes area under the curve; EAR denotes early asthmatic response,FEV₁ denotes forced expiratory volume in 1 second; FEV₁-time AUC denotesdenotes area under the curve of the time- adjusted FEV₁; % FEV₁-time AUCdenotes area under the curve of the time- adjusted area under thepercent fall curve in FEV₁; LAR denotes late asthmatic response; maximum% fall in FEV₁ denotes maximum percent fall in FEV₁ (%); MethacholinePC20 denotes the provocative concentration of methacholine causing a 20%fall in FEV₁. *P≤0.05.

FIGS. 3A to 3C show changes in peripheral blood and sputum eosinophilsand the fraction of exhaled nitric oxide. Peripheral blood eosinophils(FIG. 3A), sputum eosinophils (FIG. 3B) and fractional exhaled nitricoxide (FIG. 3C) were measured from pre-dosing baseline on day −15 untilday 85. Data are shown as mean ±SEM. FEeNO denotes fractional exhalednitric oxide, red arrows denote dosing, black arrows denote allergeninhalation challenge, * denotes p<0.05 AMG 157 compared to placebo.

FIG. 4 shows the pharmacological skewing away from the Th2 phenotype inasthma patients receiving anti-TSLP therapy.

FIGS. 5A to 5B show the cytokine profile of cells taken from an SCLEpatient when unstimulated (FIG. 5A) and stimulated (FIG. 5B) withPMA/ionomycin.

DETAILED DESCRIPTION

Th2 cells are strongly implicated as contributors to establishment andprogression of allergic asthma. In the clinical trial settingunderstanding if blocking a therapeutic target leads to changes in theTh2 cells or the Th2/Th1 ratio may be a useful exploratory objective.Moreover, the relative proportion of polarized helper T cells atbaseline may help to match patients with optimal treatments. Similarly,in the cancer setting levels of Th subsets (in particular Th1, Th17 andTreg) and functionality of CTL may be useful metrics for identifying andoptimizing treatment regimens. The present disclosure describes an assayto measure the change in T helper cells and CTLs developed using broadstimulation with PMA and ionomycin followed by intracellular cytokinestaining to analyze the Th cytokine responses. Upon comparing a combinedmethod of Th2 cytokine staining using IL-4, IL-5, and IL-13 togetherwith an alternate method staining each cytokine separately, a combinedmethod to improve the detection of Th2 cells was developed.

Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow.

As used in the specification and the appended claims, the indefinitearticles “a” and “an” and the definite article “the” include plural aswell as singular referents unless the context clearly dictatesotherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. The followingreferences provide one of skill with a general definition of many of theterms used in this disclosure include, but are not limited to: Singletonet al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d Ed. 1994);THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker Ed., 1988);THE GLOSSARY OF GENETICS, 5th Ed., R. Rieger et al. (Eds.), SpringerVerlag (1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY OFBIOLOGY (1991).

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Wheneverthe term “about” or “approximately” precedes the first numerical valuein a series of two or more numerical values, it is understood that theterm “about” or “approximately” applies to each one of the numericalvalues in that series.

The term “cytokine” as used herein refers to one or more small (5-20 kD)proteins released by cells that have a specific effect on interactionsand communications between cells or on the behavior of cells, such asimmune cell proliferation and differentiation. Functions of cytokines inthe immune system include, promoting influx of circulating leukocytesand lymphocytes into the site of immunological encounter; stimulatingthe development and proliferation of B cells, T cells, peripheral bloodmononuclear cells (PBMCs) and other immune cells; and providingantimicrobial activity. Exemplary immune cytokines, include but are notlimited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10,IL-12, IL-13, IL-15, IL17A, IL-17F, IL-18, IL-21, IL-22, interferon(including IFN alpha, beta, and gamma), tumor necrosis factor (includingTNF alpha, beta), transforming growth factor (including TGF alpha,beta), granulocyte colony stimulating factor (GCSF), granulocytemacrophage colony stimulating factor (GMCSF) and thymic stromallymphopoietin (TSLP).

A “T helper (Th) 1 cytokine” or “Th1-specific cytokine” refers tocytokines that are expressed (intracellularly and/or secreted) by Th1 Tcells, and include IFN-g, TNF-a, IL-12 and in some populations, IL-2. A“Th2 cytokine” or “Th2-specific cytokine” refers to cytokines that areexpressed (intracellularly and/or secreted) by Th2 T cells, includingIL-4, IL-5, IL-13, IL-10, and, in certain populations IL-2. A “Th17cytokine” or “Th17-specific cytokine” refers to cytokines that areexpressed (intracellularly and/or secreted) by Th17 T cells, includingIL-17A, IL-17F, IL-22 and IL-21. Certain populations of Th17 cellsexpress IFN-g and/or IL-2 in addition to the Th17 cytokines listedherein. A polyfunctional CTL cytokine includes IFN-g, TNF-a, IL-2 andIL-17.

The term “fluorophore” refers to a small molecule dye or protein thataccepts light energy at a given wavelength (excitation) and re-emits itat a longer wavelength (emission). In the present methods, fluorophoresare attached to a specific binding agent, such as an antibody or enzymesubstrate, and in certain embodiments detected by passing the labeledsubstance through a laser that causes excitation of the dye. Exemplaryfluorophores useful in the present methods, include but are not limitedto, Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor®488, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor®555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor®633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor®680, Alexa Fluor® 700, BODIPY FL, BODIPY 630/650, Cy2, Cy3, Cy3B, Cy3.5,CyS, Cy5.5, Cy7, ECD, FITC, FluorX®, Cascade® Blue, Pacific Blue®,Pacific Green®, Pacific Orange®, eFluor® 450, eFluor® 605NC, eFluor®625NC, eFluor® 650NC, eFluor® 660, eFluor® 710, Brilliant Violet™ (BV)fluorophores BV421, BV510, BV570, BV 605, BV650, BD HorizonTM V450, BDHorizon™ V500, Texas Red, rhodamine, cyanine, phycoerythrin (PE),phycocyanin, allophycocyanin (APC), o-phthaldehyde, fluorescamine,Oregon Green® 488, PE-APC, PE-Cy5, PerCP, PE-TR, rhodamine green andrhodol green, and tandem dyes thereof.

The terms “first fluorophore” and “second fluorophore” refer tofluorophores that are useful in the method of the disclosure and areused to designate use of fluorophores that emit at different wavelengthsand do not overlap in the method. For example, a “first fluorophore”refers to one or more fluorophores that are used to label one or morespecific binding agents in the method and emit light at a firstwavelength, and includes use of two different fluorophores that haveoverlapping spectral emission profiles within a first wavelength range.A “second fluorophore” refers to one or more fluorophore used in themethod in which the wavelength emission of the fluorophore(s) does notoverlap with the emission wavelength of the first fluorophore(s). It isalso possible to carry out the method using a “third fluorophore” orfurther subsequent fluorophore, which includes one or more dyes thatemits light at a third wavelength, or common range of wavelengths, butdoes not overlap spectrally with either the first fluoophore or thesecond fluorophore. Examples of fluorophores with overlapping spectralprofiles include FITC and Alexa Fluor 488; APC, eFluor® 660 and AlexaFluor® 647; PE and PE-Cy5; Alexa Fluor® 555 and Cy3; Alexa Fluor® 647and Cy5; and others determined to be in overlapping emissions spectra.One of skill can determine whether fluorophores have overlapping spectrausing commonly available resources that describe the emission spectra offluorophores.

The term “specifically binds” is “antigen specific”, is “specific for”,“selective binding agent”, “specific binding agent”, “antigen target” oris “immunoreactive” with an antigen refers to an antibody or polypeptidethat binds an target antigen with greater affinity than other antigensof similar sequence. It is contemplated herein that the agentspecifically binds target proteins useful in identifying immune celltypes, for example, a surface antigen (e.g., T cell receptor, CD3), acytokine (e.g., TSLP, IL-4, IL-5, IL-13, IL-17, IFN-g, TNF-a) and thelike.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, tetrameric antibodies, monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), antibody fragments that can bind an antigen (e.g., Fab′,F′(ab)2, Fv, single chain antibodies, diabodies), and recombinantpeptides comprising the forgoing as long as they exhibit the desiredbiological activity. An “immunoglobulin” or “tetrameric antibody” is atetrameric glycoprotein that consists of two heavy chains and two lightchains, each comprising a variable region and a constant region.Antigen-binding portions may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies. Antibodyfragments or antigen-binding portions include, inter alia, Fab, Fab′,F(ab′)2, Fv, domain antibody (dAb), complementarity determining region(CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv),single chain antibody fragments, chimeric antibodies, diabodies,triabodies, tetrabodies, minibody, linear antibody; chelatingrecombinant antibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), an antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or a variant or a derivative thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as one, two,three, four, five or six CDR sequences, as long as the antibody retainsthe desired biological activity.

“Monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts.

The term “sample” or “biological sample” refers to a specimen obtainedfrom a subject for use in the present methods, and includes urine, wholeblood, plasma, serum, saliva, tissue biopsies, cerebrospinal fluid,peripheral blood mononuclear cells with in vitro stimulation, peripheralblood mononuclear cells without in vitro stimulation, gut lymphoidtissues with in vitro stimulation, gut lymphoid tissues without in vitrostimulation, gut lavage, bronchioalveolar lavage, nasal lavage, andinduced sputum.

The term “fibroproliferative disease” or “fibrotic disease or disorder”refers to conditions involving fibrosis in one or more tissues. As usedherein the term “fibrosis” refers to the formation of fibrous tissue asa reparative or reactive process, rather than as a normal constituent ofan organ or tissue.

Fibrotic disorders include, but are not limited to, systemic and localscleroderma, keloids and hypertrophic scars, atherosclerosis,restenosis, pulmonary inflammation and fibrosis, interstitial lungdisease, idiopathic pulmonary fibrosis, liver cirrhosis, fibrosis as aresult of chronic hepatitis B or C infection, radiation-inducedfibrosis, fibrosis arising from wound healing, kidney disease, heartdisease resulting from scar tissue, and eye diseases such as maculardegeneration, and retinal and vitreal retinopathy. Additional fibroticdiseases include fibrosis resulting from chemotherapeutic drugs,radiation-induced fibrosis, and injuries and burns.

The terms “treat”, “treating” and “treatment” refer to eliminating,reducing, suppressing or ameliorating, either temporarily orpermanently, either partially or completely, a clinical symptom,manifestation or progression of an event, disease or conditionassociated with an inflammatory disorder described herein. As isrecognized in the pertinent field, drugs employed as therapeutic agentsmay reduce the severity of a given disease state, but need not abolishevery manifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method for determining theefficacy of treatment comprising administering to a patient therapeuticagent in an amount and for a time sufficient to induce a sustainedimprovement over baseline of an indicator that reflects the severity ofthe particular disorder.

The term “therapeutically effective amount” refers to an amount oftherapeutic agent that is effective to ameliorate or lessen symptoms orsigns of disease associated with a disease or disorder.

Specific Binding Agents

Specific binding agents such as antibodies and antibody fragments thatbind to their target antigen, e.g., a surface molecule, a cytokine andthe like, are useful in the method of the invention. In one embodiment,the specific binding agent is an antibody. The antibodies may bepolyclonal including monospecific polyclonal; monoclonal (MAbs);recombinant; chimeric; humanized, such as complementarity-determiningregion (CDR)-grafted; human; single chain; and/or bispecific; as well asfragments; variants; or derivatives thereof. Antibody fragments includethose portions of the antibody that bind to an epitope on thepolypeptide of interest. Examples of such fragments include Fab andF(ab') fragments generated by enzymatic cleavage of full-lengthantibodies. Other binding fragments include those generated byrecombinant DNA techniques, such as the expression of recombinantplasmids containing nucleic acid sequences encoding antibody variableregions.

Monoclonal antibodies may be modified for use as therapeutics ordiagnostics. One embodiment is a “chimeric” antibody in which a portionof the heavy (H) and/or light (L) chain is identical with or homologousto a corresponding sequence in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is/are identical with or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985,Proc. Natl. Acad. Sci. 81:6851-55.

In another embodiment, a monoclonal antibody is a “humanized” antibody.Methods for humanizing non-human antibodies are well known in the art.See U.S. Pat. Nos. 5,585,089 and 5,693,762. Generally, a humanizedantibody has one or more amino acid residues introduced into it from asource that is non-human. Humanization can be performed, for example,using methods described in the art (Jones et al., 1986, Nature321:522-25; Riechmann et al., 1998, Nature 332:323-27; Verhoeyen et al.,1988, Science 239:1534-36), by substituting at least a portion of arodent complementarity-determining region for the corresponding regionsof a human antibody.

Also encompassed by the invention are human antibodies that bindantigens of interest. Using transgenic animals (e.g., mice) that arecapable of producing a repertoire of human antibodies in the absence ofendogenous immunoglobulin production such antibodies are produced byimmunization with a polypeptide antigen (i.e., having at least 6contiguous amino acids), optionally conjugated to a carrier. See, e.g.,Jakobovits et al., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovitset al., 1993, Nature 362:255-58; Bruggermann et al., 1993, Year inImmuno. 7:33. See also PCT App. Nos. PCT/US96/05928 and PCT/US93/06926.Additional methods are described in U.S. Pat. No. 5,545,807, PCT App.Nos. PCT/US91/245 and PCT/GB89/01207, and in European Patent Nos.546073B1 and 546073A1. Human antibodies can also be produced by theexpression of recombinant DNA in host cells or by expression inhybridoma cells as described herein.

Human antibodies can also be produced from phage-display libraries(Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J.Mol. Biol. 222:581). These processes mimic immune selection through thedisplay of antibody repertoires on the surface of filamentousbacteriophage, and subsequent selection of phage by their binding to anantigen of choice. One such technique is described in PCT App. No.PCT/US98/17364, which describes the isolation of high affinity andfunctional agonistic antibodies for MPL- and msk-receptors using such anapproach.

Chimeric, CDR grafted, and humanized antibodies are typically producedby recombinant methods. Nucleic acids encoding the antibodies areintroduced into host cells and expressed using materials and proceduresdescribed herein. In a preferred embodiment, the antibodies are producedin mammalian host cells, such as CHO cells. Monoclonal (e.g., human)antibodies may be produced by the expression of recombinant DNA in hostcells or by expression in hybridoma cells as described herein.

Specific binding agents that bind cytokines useful for detecting T cellpopulations are contemplated herein, including but not limited to,binding agents specific for IL-4, IL-5, IL-13, IFN-g, TNF-a, IL-2,IL-17A, IL-17F, IL-22, and IL-10.

Specific binding agents that bind to cell surface markers arecontemplated for use in identification of T cell subpopulations.Exemplary cell surface markers include, but are not limited to, ST2,CRTH2, and CCR4 for Th2 cells, CXCR3 for Th1 cells, and/or CCR6 for Th17cells. T cell subpopulations can also be distinguished based onbiomarkers indicative of activation state, including PD1, CTLA4, CD4OL,ICOS, OX40, 41BB, TIM-3, GITR, HLA-DR and Ki67 of Th1, Th2, and Th17cells.

Therapeutic Agents

The methods described herein are useful in conjunction with therapeuticagents. Particularly preferred therapeutic agents include antibodiesthat antagonize cytokines or bind receptors that alter T helper cell orCTL ratios in a patient. Examples include, but are not limited to,antibodies to TSLP, TSLP receptor, IL-25, IL-17A, IL-17-B, IL-17C,IL-17D, IL-17E (aka IL-25), IL-17-F, IL-17RA, IL-17-RB, IL-17RC,IL-17RD, IL-17RE, IL-33, ST2, IL-4, IL-13, IL4/13R, IL-5, IL-5R, TNF,TNF-R, IL-6, IL-6R, IL-10, IL-10R, IL-12p35, IL-12p40, IL-18, IL-18R,IL-22, IL-23/p19, TGF-b, IL-2, IFN-g, IFN-a, IL-1, IL-1R, IL-9, IL-36and GM-CSF. Also contemplated are chemokines that are chemoattractantfor Th cells or CTLs, including but not limited to, CCL17, CCL22, CCL20,IP-10 and others.

Examples of anti-TSLP antibodies that may be used in certain embodimentsinclude, but are not limited to, those described in U.S. Pat. No.7,982,016, US Publ. Pat. Appl. No. 20090186022, and U.S. Pat. No.8,232,372. Examples of anti-TSLP receptor antibodies include, but arenot limited to, those described in U.S. Pat. No. 8,101,182. Inparticularly preferred embodiments, the anti-TSLP antibody is theantibody designated as A5 within U.S. Pat. No. 7,982,016.

Examples of anti-IL-17RA antibodies that may be used in certainembodiments include, but are not limited to, those described in U.S.Pat. Nos. 7,767,206, 7,786,234, 7,939,070, 7,833,527, 8,435,518,8,545,842, and US Publ. Pat. Appl. No. 20 130022621. Also suitable foruse in certain embodiments is the anti-IL-17RA antibody brodalumab asdescribed in Recommended International Nonproprietary Names: List 67(WHO Drug Information, Vol. 26, No. 1, 2012; World Health Organization).Examples of anti-IL-17A antibodies that can be used in certainembodiments include, but are not limited to, perakizumab as described inRecommended International Nonproprietary Names: List 69 (WHO DrugInformation Vol. 27, No. 1, 2013; World Health Organization),secukinumab as described in Proposed International Nonproprietary Names:List 102 (WHO Drug Information, Vol. 23, No. 4, 2009; World HealthOrganization), ixekizumab as described in Proposed InternationalNonproprietary Names: List 105 (WHO Drug Information, Vol. 25, No. 2,2011; World Health Organization), and tildakizumab as described inRecommended International Nonproprietary Names: List 70 (WHO DrugInformation, Vol. 27, No. 3, 2013; World Health Organization).

Examples of anti-ST2 antibodies that may be used in certain embodimentsinclude, but are not limited to, those described in WO2013173761.Particularly preferred are antibodies described as Ab1, Ab2, Ab3, Ab4,Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab30, Ab32, and Ab33 therein.

It is contemplated that essentially any antibody may be incorporatedinto the methods described herein. Exemplary antibodies (and the antigento which they specifically bind) include, but are not limited to, thosedescribed in U.S. Pat. No. 7,947,809 and U.S. Patent ApplicationPublication No. 20090041784 (glucagon receptor), U.S. Pat. Nos.7,939,070, 7,833,527, 7,767,206, and 7,786,284 (IL-17 receptor A), U.S.Pat. Nos. 7,872,106 and 7,592,429 (Sclerostin), U.S. Pat. Nos.7,871,611, 7,815,907, 7,037,498, 7,700,742, and U.S. Patent ApplicationPublication No. 20100255538 (IGF-1 receptor), U.S. Pat. No. 7,868,140(B7RP1), U.S. Pat. No. 7,807,159 and U.S. Patent Application PublicationNo. 20110091455 (myostatin), U.S. Pat. No. 7,736,644, U.S. Pat. Nos.7,628,986, 7,524,496, and U.S. Patent Application Publication No.20100111979 (deletion mutants of epidermal growth factor receptor), U.S.Pat. No. 7,728,110 (SARS coronavirus), U.S. Pat. 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No. 6,849,450(metalloproteinase inhibitor), U.S. Pat. No. 6,596,852 (LERK-5), U.S.Pat. No. 6,232,447 (LERK-6), U.S. Pat. No. 6,500,429 (brain-derivedneurotrophic factor), U.S. Pat. No. 6,184,359 (epithelium-derived T-cellfactor), U.S. Pat. No. 6,143,874 (neurotrophic factor NNT-1), U.S.Patent Application Publication No. 20110027287 (PROPROTEIN CONVERTASESUBTILISIN KEXIN TYPE 9 (PCSK9)), U.S. Patent Application PublicationNo. 20110014201 (IL-18 RECEPTOR), and U.S. Patent ApplicationPublication No. 20090155164 (C-FMS). The above patents and publishedpatent applications are incorporated herein by reference in theirentirety for purposes of their disclosure of antibody polypeptides,antibody encoding nucleic acids, host cells, vectors, methods of makingantibodies, pharmaceutical compositions, and methods of treatingdiseases associated with the respective target of the antibody.

Therapeutic Indications

The method of the invention is useful to detect T cell subsets, such asTh1, Th2 and Th17 cells, in subjects having a broad range of disease ordisorders that have an immune component, i.e., involvement of immunecells in development or progression of the disease or disorder.

Asthma: Asthma is a chronic inflammatory disorder of the airways. Eachyear, asthma accounts for an estimated 1.1 million outpatient visits,1.6 million emergency room visits, 444,000 hospitalizations (Defranceset al, 2008) Available at:http://www.cdc.gov/nchs/data/nhsr/nhsr005.pdf, and 3,500 deaths in theU.S. In susceptible individuals, asthmatic inflammation causes recurrentepisodes of wheezing, breathlessness, chest tightness, and cough. Theetiology of asthma is thought to be multi-factorial, influenced by bothgenetic environmental mechanisms,^(1,2) with environmental allergens animportant cause.^(2,3) The majority of cases arise when a person becomeshypersensitive to allergens (atopy). Atopy is characterized by anincrease in Th2 cells and Thh2 cytokine expression and IgE production.Approximately 10 million patients in the United States are thought tohave allergy-induced asthma. Despite the available therapeutic options,asthma continues to be a major health problem. Worldwide, asthmacurrently affects approximately 300 million people; by 2020, asthma isexpected to affect 400 million people (Partridge, Eur Resp Rev.16:67-72, 2007).

Allergen inhalation by atopic asthmatics induces some of themanifestations of asthma, including reversible airflow obstruction,airway hyperresponsiveness, and eosinophilic and basophilic airwayinflammation. Allergen inhalation challenge has become the predominantmodel of asthma in many species (Bates et al., Am J Physiol Lung CellMol Physiol. 297(3):L401-10, 2009; Diamant et al., J Allergy ClinImmunol. 132(5):1045-1055, 2013.)

Different asthma subtypes that are refractive to steroid treatment havebeen identified. Eosinophils are important inflammatory cells inallergic asthma that is characteristically mediated by Th2-type CD4+ Tcells. Neutrophilic airway inflammation is associated withcorticosteroid treatment in severe asthma and can be mediated by Th1- orTh17-type T cells (Mishra et al. Dis. Model. Mech. 6:877-888, 2013).

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derivedcytokine that is produced in response to pro-inflammatory stimuli anddrives allergic inflammatory responses primarily through its activity ondendritic cells (Gilliet, J Exp Med. 197:1059-1067, 2003; Soumelis, NatImmunol. 3:673-680, 2002; Reche, J Immunol. 167:336-3432001), mast cells(Allakhverdi, J Exp Med. 204:253-258, 2007) and CD34+ progenitor cells.⁹TSLP signals through a heterodimeric receptor consisting of theinterleukin (IL)-7 receptor alpha (IL-7Rα) chain and a common γchain-like receptor (TSLPR) (Pandey, Nat Immunol. 1:59-64, 2000; Park, JExp Med. 192:659-669, 2000).

Human TSLP mRNA^(10,11) and protein levels¹¹ are increased in theairways of asthmatic individuals compared to controls, and the magnitudeof this expression correlates with disease severity.¹⁰ Recent studieshave demonstrated association of a single nucleotide polymorphism in thehuman TSLP locus with protection from asthma, atopic asthma and airwayhyperresponsiveness, suggesting that differential regulation of TSLPgene expression might influence disease susceptibility.^(1,12,13) Thesedata suggest that targeting TSLP may inhibit multiple biologicalpathways involved in asthma.

Fibrotic disorders: Fibrosis is characterized by fibroblast accumulationand collagen deposition in excess of normal deposition in any particulartissue. Fibroblasts are connective tissue cells, which are dispersed inconnective tissue throughout the body. Fibroblasts secrete a nonrigidextracellular matrix containing type I and/or type III collagen. Inresponse to an injury to a tissue, nearby fibroblasts migrate into thewound, proliferate, and produce large amounts of collagenousextracellular matrix. Collagen is a fibrous protein rich in glycine andproline that is a major component of the extracellular matrix andconnective tissue, cartilage, and bone. Collagen molecules aretriple-stranded helical structures called .alpha.-chains, which arewound around each other in a ropelike helix. Collagen exists in severalforms or types; of these, type I, the most common, is found in skin,tendon, and bone; and type III is found in skin, blood vessels, andinternal organs.

Fibrotic disorders include, but are not limited to, systemic and localscleroderma, keloids and hypertrophic scars, atherosclerosis,restenosis, pulmonary inflammation and fibrosis, interstitial lungdisease, idiopathic pulmonary fibrosis, liver cirrhosis, fibrosis as aresult of chronic hepatitis B or C infection, radiation-inducedfibrosis, fibrosis arising from wound healing, kidney disease, heartdisease resulting from scar tissue, and eye diseases such as maculardegeneration, and retinal and vitreal retinopathy. Additional fibroticdiseases include fibrosis resulting from chemotherapeutic drugs,radiation-induced fibrosis, and injuries and burns.

Scleroderma: Scleroderma is a fibrotic disorder characterized by athickening and induration of the skin caused by the overproduction ofnew collagen by fibroblasts in skin and other organs. Scleroderma mayoccur as a local or systemic disease. Systemic scleroderma may affect anumber of organs. Systemic sclerosis is characterized by formation ofhyalinized and thickened collagenous fibrous tissue, with thickening ofthe skin and adhesion to underlying tissues, especially of the hands andface. The disease may also be characterized by dysphagia due to loss ofperistalsis and submucosal fibrosis of the esophagus, dyspnea due topulmonary fibrosis, myocardial fibrosis, and renal vascular changes.(Stedman's Medical Dictionary, 26.sup.th Edition, Williams & Wilkins,1995)). Pulmonary fibrosis affects 30 to 70% of scleroderma patients,often resulting in restrictive lung disease (Atamas et al. Cytokine andGrowth Factor Rev 14: 537-550 (2003)). Idiopathic pulmonary fibrosis isa chronic, progressive and usually lethal lung disorder, thought to be aconsequence of a chronic inflammatory process (Kelly et al., Curr PharmaDesign 9: 39-49 (2003)).

Lupus: Systemic Lupus Erythematosus (SLE) is an autoimmune diseasecaused by recurrent injuries to blood vessels in multiple organs,including the kidney, skin, and joints. SLE is estimated to affect over500,000 people in the United States. In patients with SLE, a faultyinteraction between T cells and B cells results in the production ofautoantibodies that attack the cell nucleus. These include anti-doublestranded DNA and anti-Sm antibodies. Autoantibodies that bindphospholipids are also found in about half of SLE patients, and areresponsible for blood vessel damage and low blood counts. Immunecomplexes accumulate the kidneys, blood vessels, and joints of SLEpatients, where they cause inflammation and tissue damage. It has beenhypothesized that Th2 cells in contribute to overproduction ofautoantibodies.

Lupus also includes subacute cutaneous lupus (SCLE). Other disorders inwhich autoantibodies play a role include, Sjogren's syndrome and Immunethrombocytopenic purpura (ITP).

Cancer: Recent research has shown that many cancers have an immunecomponent that contributes to both progression of disease as well askilling of tumor cells. Certain cancers have been associated withskewing toward a Th1 or Th2 phenotype. Th1-like cells are typicallyinvolved in promoting cell-mediated immunity, initiating a cytotoxicresponse and generally are considered one of the host's importantanti-cancer mechanism. It has been shown that the Th1 cytokine IFN-g canpromote anti-tumor activity in vivo (Ikeda et al., Cytokine GrowthFactor Rev. 13:95-109, 2002). Ito et al. (Anticancer Research25:2027-2031, 2005) describe that in non-small cell lung carcinomapatients, subjects with a low Th1/Th2 ratio in peripheral blood had asignificantly better prognosis than those with a high Th1/Th2 ratio inStage II or III cancer. Dai et al. (J Immunother. 36(4):248-57, 2013)demonstrated that clearance of ovarian tumors and melanoma tumors in amouse model was associated with a shift from a Th2 environment to acytotoxic Th1 environment. Additionally, Th17 cells have been shown tobe cytotoxic for tumor cells in certain studies, but also can beindicators of unfavorable outcomes in other studies (Muranski et al.,Blood 121:2402-2414, 2013). The methods herein are useful to identifythe Th population skewing in cancer before and after treatment and toalter administration of cancer therapeutics in order to skew the Th1population to one which will promote regression of tumors in a subject.

Exemplary cancers contemplated herein include, but are not limited to,adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma,anal cancer, anorectal cancer, cancer of the anal canal, appendixcancer, childhood cerebellar astrocytoma, childhood cerebralastrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliarycancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer,bladder cancer, urinary bladder cancer, bone and joint cancer,osteosarcoma and malignant fibrous histiocytoma, brain cancer, braintumor, brain stem glioma, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodeimal tumors, visual pathway andhypothalamic glioma, breast cancer, bronchial adenomas/carcinoids,carcinoid tumor, gastrointestinal, nervous system cancer, nervous systemlymphoma, central nervous system cancer, central nervous systemlymphoma, cervical cancer, childhood cancers, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma,lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrialcancer, esophageal cancer, extracranial germ cell tumor, extragonadalgerm cell tumor, extrahepatic bile duct cancer, eye cancer, intraocularmelanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, ovarian germ cell tumor, gestational trophoblastictumor glioma, head and neck cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocularcancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidneycancer, renal cancer, kidney cancer, laryngeal cancer, acutelymphoblastic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, hairy cell leukemia, lip andoral cavity cancer, liver cancer, lung cancer, non-small cell lungcancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkinlymphoma, primary central nervous system lymphoma, Waldenstrammacroglobulinemia, medulloblastoma, melanoma, intraocular (eye)melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma,metastatic squamous neck cancer, mouth cancer, cancer of the tongue,multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterinecancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer(melanoma), merkel cell skin carcinoma, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter and otherurinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor. In some embodiments,the tumor is associated with a cancer selected from the group consistingof breast cancer, melanoma, prostate cancer, pancreatic cancer, head andneck cancer, lung cancer, non small-cell lung carcinoma, renal cancer,colorectal cancer, colon cancer, ovarian cancer, liver cancer andgastric cancer. In some embodiments, the cancer is pancreatic cancer.

Additional diseases, disorders, or conditions contemplated by the methodherein include, but are not limited to, inflammation, autoimmunedisease, cartilage inflammation, fibrotic disease and/or bonedegradation, arthritis, rheumatoid arthritis, juvenile arthritis,juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoidarthritis, polyarticular juvenile rheumatoid arthritis, systemic onsetjuvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenileenteropathic arthritis, juvenile reactive arthritis, juvenile Reiter'sSyndrome, SEA Syndrome (Seronegativity, Enthesopathy, ArthropathySyndrome), juvenile dermatomyositis, juvenile psoriatic arthritis,juvenile scleroderma, juvenile systemic lupus erythematosus, juvenilevasculitis, pauciarticular rheumatoid arthritis, polyarticularrheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosingspondylitis, enteropathic arthritis, reactive arthritis, Reiter'sSyndrome, SEA Syndrome (Seronegativity, Enthesopathy, ArthropathySyndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemiclupus erythematosus, vasculitis, myolitis, polymyolitis,dermatomyolitis, osteoarthritis, polyarteritis nodossa, Wegener'sgranulomatosis, arteritis, ploymyalgia rheumatica, sarcoidosis,scleroderma, sclerosis, primary biliary sclerosis, sclerosingcholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttatepsoriasis, inverse psoriasis, pustular psoriasis, erythrodermicpsoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still'sdisease, Systemic Lupus Erythematosus (SLE), multiple sclerosis,myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease,ulcerative colitis, celiac disease, multiple schlerosis (MS), asthma,COPD, Guillain-Barre disease, Type I diabetes mellitus, Graves' disease,Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, andthe like.

Labels

In some embodiments, the antibody substance is labeled to facilitate itsdetection. A “label”, “detectable label” or a “detectable moiety” is acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, labelssuitable for use in the present disclosure include, radioactive labels(e.g., 32P), fluorophores (e.g., fluorescein), electron dense reagents,enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, orhaptens as well as proteins which can be made detectable, e.g., byincorporating a radiolabel into the hapten or peptide, or used to detectantibodies specifically reactive with the hapten or peptide.

Examples of labels include, but are not limited to, fluorescent dyesdescribed herein, radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P),enzymes (e.g., horse radish peroxidase, alkaline phosphatase and otherscommonly used in an ELISA), and colorimetric labels such as colloidalgold, colored glass or plastic beads (e.g., polystyrene, polypropylene,latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. Preferably, thelabel in one embodiment is covalently bound to the biopolymer using anisocyanate reagent for conjugation of an active agent according to thedisclosure. In one aspect of the present disclosure, the bifunctionalisocyanate reagents of the disclosure can be used to conjugate a labelto a biopolymer to form a label biopolymer conjugate without an activeagent attached thereto. The label biopolymer conjugate may be used as anintermediate for the synthesis of a labeled conjugate according to thedisclosure or may be used to detect the biopolymer conjugate. Asindicated above, a wide variety of labels can be used, with the choiceof label depending on sensitivity required, ease of conjugation with thedesired component of the assay, stability requirements, availableinstrumentation, and disposal provisions. Non-radioactive labels areoften attached by indirect means. Generally, a ligand molecule (e.g.,biotin) is covalently bound to the molecule. The ligand then binds toanother molecules (e.g., streptavidin) molecule, which is eitherinherently detectable or covalently bound to a signal system, such as adetectable enzyme, a fluorescent compound, or a chemiluminescentcompound.

The compounds of the present disclosure can also be conjugated directlyto signal-generating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes suitable for use as labels include, but are notlimited to, hydrolases, particularly phosphatases, esterases andglycosidases, or oxidotases, particularly peroxidases. Fluorescentcompounds, i.e., fluorophores, suitable for use as labels include, butare not limited to, fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, etc. Further examples of suitablefluorophores include, but are not limited to, eosin, TRITC-amine,quinine, fluorescein W, acridine yellow, lissamine rhodamine, B sulfonylchloride erythroscein, ruthenium (tris, bipyridinium), Texas Red,nicotinamide adenine dinucleotide, flavin adenine dinucleotide, etc.Chemiluminescent compounds suitable for use as labels include, but arenot limited to, luciferin and 2,3-dihydrophthalazinediones, e.g.,luminol. For a review of various labeling or signal producing systemsthat can be used in the methods of the present disclosure, see U.S. Pat.No. 4,391,904.

Methods

The present disclosure provides a whole blood stimulation method thatallows measurement of cell-signaling changes in T cell populations, suchas Th1/Th2/Th17/Th22 cell and/or CTL subsets, in the context of therapywith a therapeutic agent. For example, the disclosure demonstratesskewing away from a Th2 profile in asthmatics treated with anti-TSLPneutralizing antibody. Further, determining the cytokine expressionprofile at baseline is useful to characterize a sub-population ofpatients receiving treatment and adjust levels of therapeuticadministration for patients. This assay is useful to evaluate T cellsubsets in clinical trials in multiple inflammatory diseases. Thisspecific application has not previously been demonstrated in humanclinical trials.

The methods herein provide for detecting a ratio of Th2 and Th1 cells ina sample from a patient comprising measuring levels of Th2-specific andTh1-specific cytokines, the method comprising a) contacting the samplewith i) two or more specific binding agents that bind two or more Th2cytokines, wherein the two or more specific binding agents for theTh2-specific cytokines are labeled with a first fluorophore; and ii) atleast one specific binding agent that binds a Th1 cytokine, wherein thespecific binding agent for the Th1 cytokine is labeled with a secondfluorophore that is different from the first fluorophore; b) measuringlevels of the first and second fluorophores in the sample anddesignating the cell as a Th1 or Th2 cell based on the level ofTh1-specific and Th2-specific fluorophore detected; and c) determiningthe ratio of Th2 to Th1 cells in a sample. The method is optionallycarried out to determine the ratio of Th17 cells, wherein the Th17cytokines are labeled with a third fluorophore as described herein.

The method is also adapted to determine the ratio of Th1/Th17 cells andTh2/Th17 cells. It is also contemplated that the converse ratio, i.e.,reverse of the numerator and denominator for a stated ratio herein, iscalculated using the method above.

In various embodiments, the Th2 cytokines are selected from the groupconsisting of IL-4, IL-5 and IL-13. In some embodiments, the Th1cytokines are selected from the group consisting of interferon gamma(IFN-g), tumor necrosis factor alpha (TNF-a) and IL-2. In variousembodiments, the Th17 cytokines are selected from the group consistingof IL-17A, IL-17F, IFN-g and IL-22. In various embodiments, the specificbinding agent is an antibody specific for a Th1-specific, Th2-specificor Th17-specific cytokine.

Also contemplated are methods of detecting CD8+ CTLs. The presentdisclosure provides a method for detecting a subset of CTL, such aspolyfunctional CTLs, the method comprising a) contacting the sample withone or more specific binding agents that bind one or more polyfunctionalCTL cytokines, wherein the one or more specific binding agents for thepolyfunctional CTL cytokines are labeled with a first fluorophore and/orsecond fluorophore and/or third fluorophore; b) measuring levels of thefirst and second and/or third fluorophores in the sample and designatingthe cell as a polyfunctional CTL cell based on the level of specificfluorophore detected; and c) determining the ratio of polyfunctional CTLto other T cells, e.g., Th, Treg, NKT or gdT, in a sample.

In various embodiments, polyfunctional CTL cytokines are selected fromthe group consisting of IFN-g, IL-17, TNF-a and IL-2.

In one embodiment the cytokines are contacted intracellularly.Contacting intracellulary is carried out following protocols asdescribed in the art for intracellular staining for flow cytometry (Seee.g., Warrington et al., Arthritis Res Ther. 8(4):R136, 2006; Ito etal., Anticancer Research 25:2027-2031, 2005). Briefly, cells areobtained from a sample and fixed and permeabilized, e.g., using afixation permeabilization buffer. The cells are then contacting thecells with a specific binding agent that binds a target antigen such asa cytokine. The cells are optionally stimulated in vitro usingPMA/ionomycin or another stimulant prior to fixation as described in theExamples.

In some embodiments, the cytokines are contacted extracellularly. Incertain embodiments, the cytokines are secreted from the cells in vitroor in are detectable in fluid samples. The sample fluid can then becontacted with specific binding agents that bind a cytokine of interestand the level of cytokine determined, wherein the level of cytokine ispredictive of the skewing to the particular Th1 , Th2 or Th17 phenotypeor CTL population in the subject sample. If cells are stimulated invitro, cytokines excreted into the fluid could be measured.

It is contemplated that the sample is obtained at various times beforeand after administration of a therapeutic agent. Obtaining a sampleconcurrent with administration of a therapeutic agent does not requirethat the agent be administered at the same time as obtaining the sample,as long as there is an overlap in the time period during which theagents are exerting their therapeutic effect. Simultaneous or sequentialtiming is contemplated.

In certain embodiments, the relative number (percentage) of Th1, Th2,T17 cells or CTLs is compared at various times before and afteradministration of a therapeutic agent. In certain embodiments,administration of the therapeutic agent causes a decrease in therelative number of Th2 cells present in samples collected from asubject.

It is contemplated that the agent is administered concurrently withobtaining a sample for analysis, with concurrently referring to agentsgiven within 30 minutes before or after obtaining the sample.

In another aspect, the therapeutic agent is administered prior toobtaining the sample. Prior administration refers to administration ofthe agent within the range of one week prior to obtaining the sample, upto 30 minutes before obtaining the sample. It is further contemplatedthat the agent is administered subsequent to obtaining the sample.Subsequent administration is meant to describe administration from 30minutes after treatment up to one week after therapeutic administration.

In certain embodiments, the therapeutic agent is administered for oneweek, two weeks, three weeks, four weeks, six weeks, two months, threemonths or more prior to obtaining the sample.

In various embodiments, the asthma patient has atopic asthma or mildasthma. In certain embodiments, the Th2/Th1 ratio is skewed toward theTh2 phenotype and this skewing could identify the patient as a candidatefor treatment with a therapeutic agent that targets the Th2 pathway. Invarious embodiments, the patient may be a candidate for Th2 targetedtherapy if the ratio of Th2/Th1 cells is approximately 0.2 or higher.

In certain embodiments, the patient is identified as responsive totreatment of asthma if the ratio of Th2/Th1 cells decreases by 20%, 30%,40%, 50%, 60% or more. In various embodiments, the ratio of Th2/Th1cells decreases by 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85% or 90%, or within the range of 20-40%, 40-60%, 60-80%or 50-90%. In one embodiment, a Th2/Th1 ratio at which the patient isidentified as responsive to treatment is approximately 0.1 or below.

In various embodiments, the disclosure provides a method of altering thedose regimen of an anti-TSLP agent in treating an immune disordercomprising determining the ratio of Th2/Th1 and/or Th17 cells in asample and altering the dose of anti-TSLP antibody if the ratio ofTh2/Th1 and/or Th17 cells changes during treatment, wherein the dose oftherapeutic is increased if the ratio of Th2/Th1 cells is stable orincreases indicating skewing towards Th2 profile (i.e., increasing theproportion of Th2 cells and/or decreasing the proportion of Th1 cells);and wherein the dose of therapeutic is decreased if the ratio of Th2/Th1cells decreases indicating reduction in Th2 profile (i.e., decreasingthe proportion of Th2 cells and/or increasing the proportion of Th1cells). In certain embodiments, the dose regimen is not altered. Rather,the dose regimen is continued as a result of the present assay.

In one embodiment, the present method provides a validation protocol fora 7-color intracellular cytokine assay for Th1, Th2 and Th17 cells byflow cytometry and a six color immunophenotyping panel for Th17 cellsurface markers and memory T cell subsets in whole blood. In certainembodiments, the percentage of Th2 cytokine producing cells ranges from1 to 5%. While unstimulated samples showed a background level of 0.16%it was found that pre-incubation with unlabeled detector antibodies toblock staining suggested a lower limit of 0.5%. Longitudinal sampling aswell as spike-recovery experiments using isolated CRTH2+ cells suggeststhe assay is sensitive enough to detect changes in Th2 cells over time.

In one embodiment, the assay is a whole blood flow cytometry method tomeasure cytokine production (specifically, IL-2, IL-4-5-13, 1L-17A,IL-22, IFN-y, TNF-a) from specific cell populations in human peripheralblood samples. This method was used to demonstrate reduction ofTh2-specific intracellular cytokine production and change in the Th2/Th1ratio by a TSLP-specific antibody in an inhaled allergen challenge studyin asthmatic subjects. The described assay has utility in demonstratingpharmacodynamic activity, dose selection, and patient stratification foranti-inflammatory therapies, such as anti-TSLP antibodies, and forimmune related disorders broadly.

The method is also useful to determine if skewing away from or towardTh2-specific cytokine plays a role in the efficacy of treatment, e.g.,using an anti-TSLP antibody. The method can determine if levels of IL-4,IL-5 or 11-13 are changed in the treated patient and how therapeuticadministration can be changed to maximize therapeutic benefit.

Administration and Dosing

In one aspect, methods of the present disclosure include a step ofadministering a therapeutic agent, optionally in a pharmaceuticallyacceptable carrier or excipient. In certain embodiments, thepharmaceutical composition is a sterile composition.

Administration is performed using any medically-accepted means forintroducing a therapeutic directly or indirectly into a mammaliansubject, including but not limited to injections, oral ingestion,intranasal, topical, transdermal, parenteral, inhalation spray, vaginal,or rectal administration. The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, and intracisternal injections,as well as catheter or infusion techniques. Administration by,intradermal, intramammary, intraperitoneal, intrathecal, retrobulbar,intrapulmonary injection and or surgical implantation at a particularsite is contemplated as well.

In one embodiment, administration is performed systemically or at thesite of a cancer, fibrosis or affected tissue needing treatment bydirect injection into the site or via a sustained delivery or sustainedrelease mechanism, which can deliver the formulation internally. Forexample, biodegradable microspheres or capsules or other biodegradablepolymer configurations capable of sustained delivery of a composition(e.g., a soluble polypeptide, antibody, or small molecule) can beincluded in the formulations of the disclosure implanted near or at siteof cancer, fibrosis or affected tissue or organ.

Therapeutic compositions may also be delivered to the patient atmultiple sites. The multiple administrations may be renderedsimultaneously or may be administered over a period of time. In certaincases it is beneficial to provide a continuous flow of the therapeuticcomposition. Additional therapy may be administered on a period basis,for example, hourly, daily, weekly, every 2 weeks, every 3 weeks,monthly, or at a longer interval.

Also contemplated in the present disclosure is the administration ofmultiple agents, such as an antibody composition in conjunction with asecond agent as described herein, including but not limited to a ananti-inflammatory agent, a chemotherapeutic agent or an agent useful totreat fibrosis.

The amounts of therapeutic agent, such as an antibody, in a given dosagemay vary according to the size of the individual to whom the therapy isbeing administered as well as the characteristics of the disorder beingtreated. In exemplary treatments, it may be necessary to administerabout 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day,100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 500 mg/day or 1000mg/day. These concentrations may be administered as a single dosage formor as multiple doses. Standard dose-response studies, first in animalmodels and then in clinical testing, reveal optimal dosages forparticular disease states and patient populations.

It will also be apparent that dosing may be modified if traditionaltherapeutics are administered in combination with therapeutics of thedisclosure.

Kits

As an additional aspect, the disclosure includes kits which comprise oneor more compounds or compositions packaged in a manner which facilitatestheir use to practice methods of the disclosure. In one embodiment, sucha kit includes a compound or composition described herein (e.g., acomposition comprising a specific binding agent alone or affixed to alabel or fluorophore), packaged in a container such as a sealed bottleor vessel, with a label affixed to the container or included in thepackage that describes use of the compound or composition in practicingthe method. Preferably, the compound or composition is packaged in aunit dosage form.

Additional aspects and details of the disclosure will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

EXAMPLES Example 1

Materials and Methods

Study Participants

Eligible participants were nonsmoking men and women aged 18 to 60 yearswith mild stable atopic asthma confirmed by positive skin-prick test,forced expiratory volume (FEV) in 1 second (FEV₁)≥70% of predicted, andairway hyperresponsiveness. Participants were tested out of season forpollens affecting their asthma and had no other lung disease. No asthmacontroller treatments were allowed; inhaled short-acting β₂ agonists asrescue treatments used less than twice weekly were permitted. All otherasthma medications were discontinued at least 4 weeks before enrollment.Participants were excluded for worsening of asthma, respiratory-relatedvisits to the emergency department within 6 weeks, prior use of AMG 157,or known sensitivity to any AMG 157 excipients.

Study Design and Oversight

This proof-of-concept, randomized, double-blind, placebo-controlledstudy was conducted in 5 centers in Canada. Participants were randomlyassigned by interactive voice response system 1:1 to receive 700 mg AMG157 or placebo by 1-hour intravenous infusion on study days 1, 29, and57. Allergen and methacholine inhalation challenges were performed atdays −15, −14 and −13, days 41, 42 43, 83, 84 and 85. Fractional exhalednitric oxide (F_(E)NO) levels were measured on days −15, −13, 1, 41, 43,83 and 85, induced sputum was measured on days −15, −14, −13, 1, 41, 42,43, 83, 84 and 85, and blood samples were measured on days −15, 1, 29,43, 57, 85, 113 and 169. The primary endpoint was the late asthmaticresponse (LAR) measured between 3 and 7 hours after the allergenchallenge expressed as maximum percent fall in FEV₁ and area under thecurve (AUC) of the time-adjusted percent fall in FEV₁ (% FEV1-time AUC).The secondary endpoints were the LAR measured by minimum FEV₁ and AUC ofthe time-adjusted minimum FEV₁ (FEV1-time AUC), early asthmatic response(EAR) measured between 0 and 2 hours after the allergen challenge, andthe safety, side-effect profile and immunogenicity of AMG 157.Exploratory endpoints included sputum and blood eosinophils, F_(E)NO,Th2 cytokines, Th2/Th1 cell ratio and total IgE in blood, andmethacholine PC₂₀. Safety evaluations included incidence and severity ofadverse events, changes in electrocardiogram, laboratory profiles, vitalsigns and the presence of anti-AMG 157 antibodies.

The study protocol was approved by the institutional research ethicscommittees at each participating center, and all participants providedwritten informed consent.

Laboratory Procedures

The allergen for inhalation was selected using results from skin-pricktesting. The allergen inhalation challenge was performed as described.¹⁴During a screening challenge on day −14, doubling concentrations ofallergen were inhaled over 2 minutes by tidal breathing from a Wrightnebulizer (Roxon, Quebec) filled with 2-3 mL solution, until a ≥20% fallin FEV₁ at 10 minutes post-allergen was reached. The FEV₁ was thenmeasured at regular intervals for 7 hours. The EAR (0-2 hours) and LAR(3-7 hours) endpoints were calculated. Selection of allergen dose andmethacholine challenges were performed as described.¹⁵ Venous blood wassampled for leukocytes, total IgE and cytokines, and airway eosinophilswere sampled from induced sputum using a standard method.¹⁶ F_(E)NOmeasurements followed American Thoracic Society guidelines.¹⁷

Statistical Analysis

A sample size of 30 (15 per treatment arm) participants was selectedbased upon empirical evidence from previous studies¹⁸⁻²⁰ suggesting that15 participants per treatment group would provide adequate power todifferentiate the LAR-attenuating effect of AMG 157 placebo. Theanalysis population for each endpoint included all available data fromall randomized participants who received at least 1 dose of AMG 157 orplacebo, with data analyzed in accordance with the initial treatmentreceived. The EAR and LAR were analyzed using a repeated measuresanalysis of covariance (ANCOVA) including treatment and visit asindependent variables, treatment by visit interaction term, andcorresponding pre-dose measure as a model covariate. The mean treatmentdifference, the corresponding 95% confidence interval (CI), andtwo-sided P value were estimated and reported at each visit. Theexploratory endpoints were analyzed using repeated measures ANCOVA(Supplemental Appendix).The summary data are reported as mean±SEM, lognormally distributed endpoints are presented as geometric means (95% CI)while categorical data are presented as number (%).

Results

Study Population

A total of 31 participants were randomized, with 16 assigned to AMG 157and 15 to placebo. All participants received at least one dose of studydrug in accordance with the randomization schedule. The study wascarried out for approximately 18 months. Twenty-eight participants (90%)completed the full intervention period, and 27 (87%) completed thestudy. Three of the 4 participants who did not complete the study werelost to follow-up (2 placebo, 1 AMG 157) and 1 participant withdrew atday 34 due to worsening of asthma (AMG 157). One participant from eachgroup did not complete the day 84 allergen challenge, and oneparticipant aborted the day 84 allergen challenge before LAR measurement(AMG 157). Demographics and inhaled allergens were similar in the twogroups, and there were no significant differences in any of the baselinevariables measured between the two groups.

Endpoints

AMG 157 treatment partially attenuated both the LAR and EAR relative toplacebo at days 42 and 84 in each of the 4 allergen challenge endpoints(FIG. 1, FIG. 2). A statistically significant AMG 157-associatedattenuation was achieved on the LAR minimum FEV₁ and FEV₁-time adjustedAUC_(3-7h) on day 42, and on the LAR maximum percent fall in FEV₁ andminimum FEV₁ on day 84, with no additional benefit of the lastinjection. The maximum percentage decrease in the FEV1 during the lateresponse was 34.0% smaller in the AMG-157 group than in the placebogroup on day 42 (P=0.09) and 45.9% smaller (a decrease of 11.7% vs.21.6%) on day 84 (P=0.02). Patients in the AMG-157 group, as comparedwith those in the placebo group, had a significant increase in theminimum FEV1 (P=0.01) and in the AUC of the time-adjusted minimum FEV₁(P=0.02) during the late response on day 42 and in the minimum FEV₁(P=0.01) on day 84. In addition, during the early response, the AUC ofthe time-adjusted percent decrease in the FEV₁ was significantly smallerand the AUC of the time-adjusted minimum FEV₁ significantly greater inthe AMG-157 group than in the placebo group (P=0.03 for bothcomparisons) on day 42, and the AUC of the time-adjusted percentdecrease in the FEV₁ was significantly smaller on day 84 (P=0.030)(FIGS. 1 and 2).

The mean baseline blood eosinophil counts decreased from296.5±40.02×10⁶/L at day −15 to 121.9±14.7×10⁶/L at day 29 with AMG 157,and from 281.1±57.3×10⁶/L at day −15 to 224.1±36.5×10⁶/L at day 29 withplacebo. (FIG. 3A). Blood eosinophil counts increased post-allergen ondays 43 and 85, however the levels were significantly lower with AMG 157treatment relative to placebo (overall treatment effect P=0.004).

AMG 157 treatment decreased sputum eosinophils before and after allergenchallenge. Mean pre-allergen sputum eosinophil levels were reduced from4.1±2.3% on day −15, to 0.4±0.1% on day 41 and 0.4±0.1% on day 83.Compared to placebo, AMG 157 significantly reduced pre-allergen sputumeosinophil levels over the course of the study (overall treatment effectP=0.015) (FIG. 3B), and significantly attenuated allergen-inducedchanges 24 hours post-challenge (overall treatment effect P=0.004).

F_(E)NO was elevated in both treatment groups under baseline conditions.Compared to placebo, AMG 157 treatment significantly decreased F_(E)NOthroughout the study (overall treatment effect P=0.002) andsignificantly attenuated allergen-induced changes 24 hours postchallenge (overall treatment effect P=0.02) (FIG. 3C).

Treatment with AMG 157 did not significantly change the pre-allergenFEV₁ values measured at days 41 and 83. There was a significant increasein methacholine PC₂₀ on days 83 and 85 with AMG 157 compared to placebo(p<0.05). The allergen-induced shift in methacholine PC₂₀ (day 41 to 43,and day 83 to 85) was numerically lower with AMG 157 treatment with anincrease of 0.76 and 0.49 doubling doses for AMG 157 relative toplacebo, respectively; however, this difference was not statisticallysignificant compared to placebo (FIG. 2). There was no effect of AMG 157on total IgE or the quantifiable serum markers in the HumanMAP®v.2.0panel. Interleukins 4, 5, and 13 and TNF levels were below level ofquantitation in >95% of samples.

Geometric mean Th2/Th1 ratio (95% Confidence Intervals) was measured insamples from treated patients. Treatment with AMG 157 was associatedwith a Th2/Th1 cell ratio numerically but not statistically lower thanplacebo (The p-value's from the ANOVA were 0.058 for the main effect oftreatment, and 0.367 for the treatment by time interaction). Thedecrease in Th2/Th1 ratio was driven mostly by a decrease in Th2 cells.Blood samples were collected in sodium heparin vacutainers andlaboratory procedures initiated within 24 hours of collection. Toclassify subsets of cytokine-producing cells, whole blood was firstactivated with phorbol 12-myristate 13-acetate (PMA) and ionomycin inthe presence of the protein transport inhibitor brefeldin A.Specifically, 10 uL of leukocyte activation cocktail (LAC, BDBiosciences, San Jose, Calif.) was mixed with 500 uL RPMI and thencombined 1-1 with whole blood in a 15 mL tube (BD Falcon). The LACstimulation level was chosen because it was found to be above EC90during establishment of the assay (comparing to titrated PMA/Ionomycin).Sample aliquots incubated similarly in the presence of brefeldin A (BDGolgiStop) alone in RPMI were included as negative controls. After a 4hour incubation at 37° C. and an overnight hold at 18° C., surface andintracellular staining procedures were performed.

Intracelluar cytokine analysis included IFN-γ (BD, clone 25723.11 FITC)and combined analysis of IL-4 (BD, clone 3010.211 PE), IL-5 (BD, cloneJES1-39D10 PE) and IL-13 (BD clone JES10-5A2 PE). Fluorescence data wereacquired on a validated BD FACSCanto II flow cytometer and analysis wasperformed in FCS Express software (De Novo, Los Angeles Calif.) using astandardized template. Th1 cells were identified as CD3⁺CD8⁻ T-cellsexpressing IFN-γ, but not IL-4, IL-5 or IL-13 and Th2 cells wereidentified as CD3⁺CD8⁻ T-cells expressing IL-4, IL-5 or IL-13, but notIFN-y. The key end point was the ratio of Th2 to Th1 cells before andafter AMG 157 treatment.

The percent of Th1, Th2 and ratio of Th2/Th1 cells were analyzed afterlog transformation in a baseline-adjusted mixed effects model includingpost-dose study visit, treatment group and the interaction between visitand treatment along with subject as a random factor. Baseline valueswere included as a covariate. Only Days 41 and 83 were considered forstatistical analysis in order to test for potential Th2/Th1 ratiochanges at time-points with AMG 157 exposure. An ANOVA was used toassess whether the treatment groups differed when averaged across allvisits (dose term) or at any given time (visit by treatment interactionterm). Confidence intervals in the figures have been adjusted for thenumber of means estimated (Dunn-Sidak). *P<0.05 compared to placebo.

The circulating Th2/Th1 cell ratios were reduced with AMG 157 treatmentrelative to placebo with reduction of 29% on day 41 (P=0.016) and 23% onday 83 (P=0.47) (Table 1)

TABLE 1 Th2/Th1 Cell Ratio: Intracellular cytokine assay Treatment 95%Difference Confidence Day Placebo AMG157 Estimate Intervals Day 41 0.0930.066 0.713 (0.55, 0.93) Day 83 0.084 0.073 0.866 (0.58, 1.30)

Safety

Treatment with AMG 157 was not associated with changes in measured labvalues, temperature, blood pressure, pulse or respiration. There were 12adverse events with placebo treatment and 15 adverse events with AMG157. There were no serious adverse events or deaths. One placebo-treatedparticipant and no AMG 157-treated participants tested positive foranti-AMG 157 antibodies.

Discussion

This study has demonstrated that treatment with monoclonal antibody AMG157 attenuated most measures of allergen-induced bronchoconstriction(EAR and LAR), along with markers of systemic and airway inflammation instable allergic asthmatic participants. As such, the weight of data isconsistent with the documented role of TSLP in inducing allergen-inducedairway responses in murine models.²¹ AMG 157 also reduced all of theinflammatory variables measured in the baseline assessment, for theduration of the study, including indices of airway inflammation (F_(E)NOand sputum eosinophils), as well as systemic inflammation (circulatingeosinophils). Whether the changes in eosinophils are responsible forattenuation of allergen-induced bronchoconstriction is unknown. Thisproof-of-concept study suggests that TSLP is not only a pivotal cytokinein allergen-induced airway responses, but also in causing persistingairway inflammation in patients with allergic asthma.

TSLP has been identified as a “master switch” for allergic inflammationin murine models.²² Higher levels of TSLP were produced in epithelialcells from asthmatic versus healthy individuals,¹¹ and polymorphisms inthe TSLP gene have been associated with both childhood and adultallergic asthma.^(13,23) TSLP strongly induced the expression of humanmajor histocompatibility complex I and II and co-stimulatory moleculessuch as CD40, CD80 and CD86 on myeloid dendritic cells.⁶ Induction ofTSLP preceded the infiltration of dendritic cells into the skin duringallergen-induced late cutaneous responses.²⁴ TSLP can also induce humanmast cell Th2 cytokine production.⁸ TSLP may additionally play a role invirus-mediated processes.²⁵

TSLP is thought to cause airway and blood eosinophilia in allergicasthmatics through activation of airway dendritic cells and increases inthe numbers of Th2 cells, with the production of pro-inflammatorycytokines, including interleukin-5 and interleukin-13.²¹ TSLP has alsobeen shown to influence production of interleukin-5 and interleukin-13from mast cells,⁸ CD34⁺ progenitors⁹ and, most recently, type 2 innatelymphoid cells.²⁶ Inhibition of interleukin-5 has been previously shownto prevent allergen-induced airway eosinophilia²⁷ which supports thishypothesis. Other airway epithelial-derived cytokines, particularlyinterleukin-25 and interleukin-33, have also been implicated inallergen-induced airway inflammation in murine models,²⁸ but therecurrently is no direct evidence implicating them in allergic asthma inhumans.

Epidemiological evidence supports an important role for environmentalallergens in the pathobiology of childhood asthma.²⁹ Allergen inhalationby allergic asthmatics results in many manifestations of asthma,including reversible airflow obstruction, airway hyperresponsiveness³°and eosinophilic and basophilic airway inflammation.³¹ Allergeninhalation challenge has been a valuable clinical model for the study ofthe mechanisms of allergic asthma and the evaluation of potential newtreatments.^(20,32) However, allergen inhalation is not responsible forthe development or persistence of asthma in many asthmatics, who arenon-allergic, or who are not exposed to allergens. Therefore, theimportance of TSLP in persisting airway inflammation in these patientscannot be extrapolated from the current study. However, pharmacologicalattenuation of allergen-induced airway responses in allergic asthmaticparticipants has previously been associated with effective asthmatreatments, even in non-allergic subjects.⁵

Histamine and cysteinyl leukotrienes from airway mast cells andbasophils contribute the major part of the EAR and LAR.^(33,34) The LARis also caused by the allergen-induced influx of inflammatory cells,particularly basophils and eosinophils.^(31,33) Therefore, AMG 157likely attenuates these responses through effects both on mast cellactivation and inflammatory cell recruitment.

For safety reasons and to avoid the potential modification ofallergen-induced airway responses by maintenance treatments such asinhaled corticosteroids or leukotriene receptor antagonists, this studywas conducted in stable, allergic asthmatics who were not on regularmaintenance asthma treatment with near normal baseline pulmonaryfunction. As in other studies evaluating this patientpopulation,^(20,32,33) the participants had evidence of airwayinflammation at the time of study enrolment, with increased F_(E)NO andsputum eosinophilia. The mechanism causing persistent airwayinflammation in these stable asthmatics is not known. Some may beregularly exposed to ubiquitous allergens, such as house-dust mite, butthis accounted for less than half of the participants studied (FIG. 2).Also, because the participants had near normal baseline FEV₁ values, itwas not possible to observe improvement in baseline FEV₁.

All currently available asthma treatments attenuate components ofallergen-induced airway responses. However, only inhaled corticosteroidsattenuate baseline airway levels of F_(E)NO and eosinophils,³⁵ as wellas allergen-induced increases in these parameters.³⁶ This studyindicates that targeting TSLP can reduce baseline F_(E)NO, and blood andairway eosinophilia. Some patients with severe refractory asthma havepersisting airway eosinophilia despite treatment with high-dose inhaledand oral corticosteroids. Targeting the Th2 cytokines interleukin-5,interleukin-13, and interleukin-4 has improved several asthmaparameters. Targeting interleukin-5 in patients with severe refractoryasthma reduced asthma exacerbations and allowed a reduction inmaintenance doses of oral corticosteroids.^(37,38) These studies suggestthat persisting airway eosinophilia is an important mechanism for somepatients with severe refractory asthma. Antibodies directed againstinterleukin-13 have been shown to improve lung function in asthmaticswith a “Th2 phenotype”, as indicated by elevated levels of circulatingperiostin.³⁹ In addition, an antibody directed against theinterleukin-4-receptor-α, the common component of the interleukin-4 andinterleukin-13 receptors, allowed removal of maintenance treatment witha combination of inhaled corticosteroids and long-acting β₂ agonistwithout a deterioration of asthma control.⁴⁰ The production of each ofthese pro-inflammatory cytokines may be a consequence (downstream) ofepithelial cell TSLP production and dendritic cell activation,suggesting that targeting TSLP may also provide benefit in these patientpopulations. However, further clinical studies will be needed toevaluate this potential benefit.

In summary, treatment for 12 weeks with AMG 157 reduced baseline F_(E)NOand blood and sputum eosinophils in allergic asthmatic participants.This treatment also attenuated allergen-induced changes in theseinflammatory parameters, as well as the EAR and LAR. These resultssupport further work on mechanisms of action and investigation of theclinical benefit of AMG 157 in patients with poorly controlled asthma.

Example 2

To determine the Th2/Th1 ratios in Example 1 a methodology was used inwhich intracellular cytokine staining was performed to determine theratio of cells expressing Th2 cytokines, such as IL-4, IL-5 and IL-13,compared to those expressing Th1 cytokines, IFN-g, TNF-a and/or IL-2.

In order to maximize the detection of Th2-related cytokines, each of theIL4, IL-5 and IL-13 cytokines were detected using anti-cytokineantibodies labelled with a phycoerythrin (PE) fluorophore. Theindividual and collective measurement of these cytokines was compared tothe level of IFN-g in the same cell population, which is indicative ofTh1 cells.

Percentages of cytokine producing cells are set out in Table 2.

TABLE 2 Panel Profile Mean SD Median Min Max Panel 1 IL-4-5-13+ (IFN-g−)3.4 0.5 3.6 2.7 4.0 IFN-g+ (IL-4-5-13−) 32.1 11.5 28.6 21.6 46.3 IL-17A+(IL-4-5-13−) 3.6 0.7 3.5 2.7 4.6 IL-2+ (IL-4-5-13−) 54.0 9.4 56.1 41.765.6 Panel 2 IL-4+ (IFN-g−) 4.9 0.6 4.8 4.0 5.9 IL-5+ (IFN-g−) 1.8 1.41.5 0.7 4.5 IL-13+ (IFN-g−) 2.3 0.3 2.2 2.0 2.7 IFN-g+ (IL-13−) 30.610.2 17.7 17.7 42.3

The Table is a result of six donors and the mean, max and min number ofcells provided. Th are defined in this assay as a percentage of CD3+CD8−lymphocytes expressing cytokine(s) indicated.

The data could also be expressed as numbers of cells if relativeabsolute T-cell count is determined in parallel. In certain embodiments,reference counting beads are used to determine cell counts fornormalization.

The increased signal to noise ratio (S/N) in Panel 1 suggests anadvantage of using combined measurement of IL-4-5-13 for detection ofTh2 cells. IL-4, IL-5 and IL-13 are largely expressed by the same cells,which is especially true for IL-5 and IL-13. Results of analysis are setout in Table 3. Results are also set out graphically in FIG. 4 and showa skewing away from Th2 in treated patients.

TABLE 3 Panel Profile Mean SD Median Min Max Panel 1 IL-4-5-13+ (IFN-g−)13.0 4.2 11.6 8.3 19.8 IFN-g+ (IL-4-5-13−) 177.9 79.3 169.2 100.0 285.7IL-17A+ (IL-4-5-13−) 38.8 8.6 41.8 21.5 44.5 IL-2+ (IL-4-5-13−) 41.7 8.940.3 32.3 54.3 Panel 2 IL-4 S/N (IFN-g−) 6.3 1.3 6.0 5.1 8.6 IL-5 S/N(IFN-g−) 5.3 0.4 5. 5.0 6.1 IL-13 S/N (IFN-g−) 8.1 2.1 2 6.6 12.1 IFN-g+(IL-13−) 152.9 47.4 7.2 89.2 217.6

Example 3

This present method was used to evaluate samples from psoriasis, andsubacute cutaneous lupus (SCLE) patients to look for impact on Th17cells. A SCLE sample was unique in that unstimulated controls showedhigh levels of IFN-g and TNF-a, suggesting this assay may be useful as adiagnostic tool to guide treatment for patients suffering from differenttypes of lupus-related diseases (FIG. 5).

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention.

All publications and patent documents cited in this application areincorporated by reference in their entirety to the same extent as if thecontents of each individual publication or patent document wereincorporated herein.

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1. A method for detecting a ratio of T helper 2 (Th2) and T helper 1(Th1) cells in a sample from a patient comprising measuring levels ofTh2-specific and Th1-specific cytokines, the method comprising: a)contacting the sample with i) two or more specific binding agents thatbind two or more Th2 cytokines, wherein the two or more specific bindingagents for the Th2-specific cytokines are labeled with a firstfluorophore; and ii) at least one specific binding agent that binds aTh1 cytokine, wherein the specific binding agent for the Th1 cytokine islabeled with a second fluorophore that is different from the firstfluorophore; b) measuring levels of the first and second fluorophores inthe sample and designating the cell as a Th1 or Th2 cell based on thelevel of Th1-specific and Th2-specific fluorophore detected; and c)determining the ratio of Th2 to Th1 cells in a sample.
 2. The method ofclaim 1 further comprising determining the ratio of Th1, Th2 and Th17cells in the sample, comprising a) contacting the sample with iii) atleast one specific binding agent that binds a Th17 cytokine, wherein thespecific binding agent for the Th17 cytokine is labeled with a secondfluorophore that is different from the first fluorophore or labeled witha third fluorophore. b) measuring levels of the first and secondfluorophores in the sample and designating the cell as a Th1, Th2 orTh17 cell based on the level of Th1-specific, Th2-specific orTh17-specific fluorophore detected; and c) determining the ratio of Th1to Th2 to Th17 cells in a sample.
 3. A method for detecting a ratio of Thelper 1 (Th1) and T helper 17 (Th17) cells in a sample from a patientcomprising measuring levels of Th1-specific and Th17-specific cytokines,the method comprising: a) contacting the sample with i) two or morespecific binding agents that bind two or more Th17 cytokines, whereinthe two or more specific binding agents for the Th17-specific cytokinesare labeled with a first fluorophore; and ii) at least one specificbinding agent that binds a Th1 cytokine, wherein the specific bindingagent for the Th1 cytokine is labeled with a second fluorophore that isdifferent from the first fluorophore; b) measuring levels of the firstand second fluorophores in the sample and designating the cell as a Th1or Th17 cell based on the level of Th1-specific and Th17-specificfluorophore detected; and c) determining the ratio of Th1 to Th17 cellsin a sample.
 4. A method for detecting a ratio of T helper 2 (Th2) and Thelper 17 (Th17) cells in a sample from a patient comprising measuringlevels of Th2-specific and Th17-specific cytokines, the methodcomprising: a) contacting the sample with i) two or more specificbinding agents that bind two or more Th2 cytokines, wherein the two ormore specific binding agents for the Th2-specific cytokines are labeledwith a first fluorophore; and ii) at least one specific binding agentthat binds a Th17 cytokine, wherein the specific binding agent for theTh17 cytokine is labeled with a second fluorophore that is differentfrom the first fluorophore; b) measuring levels of the first and secondfluorophores in the sample and designating the cell as a Th2 or Th17cell based on the level of Th2-specific and Th17-specific fluorophoredetected; and c) determining the ratio of Th2 to Th17 cells in a sample.5. The method of claim 1, wherein the Th2 cytokines are selected fromthe group consisting of IL-4, IL-5 and IL-13.
 6. The method of claim 1,wherein the Th1 cytokines are selected from the group consisting ofinterferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a) and IL-2.7. The method of claim 1, wherein the Th17 cytokines are selected fromthe group consisting of IL-17A, IL-17F, IFN-g and IL-22.
 8. The methodof claim 1, wherein the specific binding agent is an antibody specificfor a Th1-specific, Th2-specific or Th17-specific cytokine.
 9. Themethod of claim 1, wherein the patient is suffering from a disease ordisorder selected from the group consisting of asthma, allergicrhinosinusitis, allergic conjunctivitis, atopic dermatitis, fibroticdisorders, Systemic Lupus Erythematosus (SLE), multiple sclerosis andcancer.
 10. The method of claim 1, wherein the sample is obtained beforeand/or after treatment with a therapeutic agent.
 11. The method of claim1, wherein the therapeutic agent is an anti-TSLP antibody.
 12. Themethod of claim 1, wherein the first fluorophore is phycoerythrin (PE)and the second flurophore is fluorescein isothiocyanate (FITC).
 13. Themethod of claim 1, wherein the sample is whole blood, peripheral bloodmononuclear cells, cerebrospinal fluid, bronchioalveolar lavage, nasallavage, induced sputum or a biopsy from the patient.
 14. The method ofclaim 1, wherein the cytokines are contacted intracellularly.
 15. Amethod for identifying a sub-population of asthma patients responsive totreatment with a therapeutic agent comprising measuring the baselineratio of T helper 2 (Th2) and T helper 1 (Th1) cells in a patient sampleor changes in the ratio after administration of the therapeutic agent,the method comprising: a) contacting the sample with i) two or morespecific binding agents that bind two or more Th2-specific cytokines,wherein the two or more specific binding agents for the Th2-specificcytokines are labeled with a first fluorophore; and ii) at least onespecific binding agent that binds a Th1-specific cytokine, wherein thespecific binding agent for the Th1-specific cytokine is labeled with asecond fluorophore that is different from the first fluorophore; b)measuring levels of the first and second fluorophores in the sample anddesignating the cell as a Th1 or Th2 cell based on the level ofTh2-specific and Th1-specific fluorophore detected; and c) determiningthe ratio of Th2 to Th1 cells in a sample based on the level ofTh2-specific cytokine and Th1-specific cytokine detected, wherein thepatient is identified as responsive to the therapeutic agent if theratio of Th2 cells/Th1 cells decreases; and d) altering treatment withthe therapeutic agent if the patient is determined to be non-responsiveto the therapeutic agent or maintaining the dose of therapeutic agent ifthe patient is determined to be responsive to treatment with thetherapeutic agent.
 16. The method of claim 15, wherein the therapeuticagent is an anti-TSLP antibody.
 17. The method of claim 15, wherein thetherapeutic agent is administered for one week, two weeks, three weeks,four weeks, six weeks, two months, three months or more prior toobtaining the sample.
 18. The method of claim 15, wherein the patient isidentified as responsive to treatment if the ratio of Th2/Th1 cellsdecreases by 20%, 30%, 40%, 50%, 60% or more.
 19. The method of claim15, a Th2/Th1 ratio at which the patient is identified as responsive totreatment is approximately 0.1 or below.
 20. A method of altering thedose regimen of an anti-TSLP agent in treating an immune disordercomprising determining the ratio of Th2/Th1 and/or Th17 cells in asample using the method of claim 1 and altering the dose of anti-TSLPantibody if the ratio of Th2/Th1 and/or Th17 cells changes duringtreatment, wherein the dose of therapeutic is increased if the ratio ofTh2/Th1 cells is stable or increases indicating skewing towards Th2profile; wherein the dose of therapeutic is decreased if the ratio ofTh2/Th1 cells decreases indicating reduction in Th2 profile.
 21. Themethod of claim 20 wherein the immune disorder is selected from thegroup consisting of asthma, allergic rhinosinusitis, allergicconjunctivitis, atopic dermatitis and fibrotic disorders.
 22. A methodof altering the dose regimen of an asthma therapeutic comprisingdetermining the ratio of Th2/Th1 and/or Th17 cells in a sample using themethod of claim 1 and altering the dose of asthma therapeutic if theratio of Th2/Th1 and/or Th17 cells changes during treatment, wherein thedose of therapeutic is increased if the ratio of Th2/Th1 cells is stableor increases indicating skewing towards Th2 profile; wherein the dose oftherapeutic is decreased if the ratio of Th2/Th1 cells decreasesindicating reduction in Th2 profile.